The present invention relates to a gene associated with invasive processes, for example endometriosis, to a polypeptide encoded by it, to an antibody directed against the polypeptide, and to the pharmaceutical application of the nucleic acid, the polypeptide and the antibody.
Endometriosis is the second most common disease in women and is defined as the occurrence of endometrial cells outside the womb. Endometriosis affects about one in five women of reproductive age, and as many as one in two women with fertility problems.
In normal circumstances the endometrium is only found in the womb. In endometriosis, tissue with a histological appearance resembling the endometrium is found outside the womb, for example externally on the womb, on the intestine or even in the pancreas or the lung. Although these endometriotic foci are located outside the womb, they also bleed during menstruation, thus they are influenced by hormones of the female cycle. Since endometriotic foci like the endometrium go through volume changes during the cycle, these changes may cause pain depending on location. Moreover, the body reacts to endometriotic cells with an inflammatory response which again causes pain. Furthermore, inflammation leads to adhesions in the area of the ovaries and fallopian tubes and, as a result of these, is responsible for a so-called mechanical sterility of affected women. Apparently however, in endometriosis messengers are released as well (e.g. cytokines, prostaglandins) which can reduce the fertility of affected women even in the absence of adhesions.
In view of their pathobiological properties, endometriotic cells could be classified as being between normal cells and tumor cells: on the one hand they show no neoplastic behavior, on the other hand, however, they are, like metastasizing tumor cells, capable of moving across organ boundaries in the organism and of growing into other organs, i.e. they show invasive behavior. For this reason endometriotic cells are defined as xe2x80x9cbenign tumor cellsxe2x80x9d in the literature, although up until now no tumor-specific-mutations in proto-oncogenes have been found in cells of this type.
Since the pathogenesis of endometriosis is still not clarified completely, there are as yet no effective options for the therapy or prevention of endometriosis-associated diseases.
It was the object of the invention to identify novel genes which play a role in invasive processes and which may be associated with the pathophysiological phenotype of endometriosis.
This object is achieved according to the invention by identifying, cloning and characterizing a gene which is called an endometriosis-associated gene and which codes for a polypeptide. This gene sequence was discovered with the aid of differential display RT-PCR (Liang and Pardee, Science 257 (1992), 967-971). For this, invasive and noninvasive variants of an endometriotic cell line were compared with each other. In the process a cDNA sequence was found which is specific for the invasive variant of endometriotic cells. An associated RNA of 4 kb in length was found. A corresponding cDNA isolated from a cDNA phage bank has an open reading frame (ORF) of 302 amino acids.
The present invention relates to a nucleic acid which comprises
(a) the nucleotide sequences depicted in SEQ ID NO. 1, 3 or/and 5, a combination or a protein-encoding segment thereof,
(b) a nucleotide sequence corresponding to the sequence in (a) within the scope of the degeneracy of the genetic code or
(c) a nucleotide sequence hybridizing with the sequences in (a) and/or (b) under stringent conditions.
The nucleic acids preferably code for a polypeptide associated with invasive processes or a segment thereof.
The following nucleotide sequences have been deposited in the EMBL EST database with the following accession numbers: Z98886, Ac003017, AL023586, Aa52993, Aa452856. These sequences do not represent nucleic acids according to the invention. The first two of these sequences are DNAs which were isolated from human brain and show over 90% identical bases to SEQ. ID NO. 1 in the segments from nucleotide 970 to about 2000 and from 760 to about 1450, respectively, or in the segments from nucleotide 1054 to 2084 and from 844 to about 1534 in relation to SEQ ID NO. 3 which has 84 additional bases at the 5xe2x80x2 end. AL023586 is also a human sequence which is very similar to Z98885 and also has homology with SEQ ID NO. 1 in the region from 970 to about 2000.
Sequences Aa452993 and Aa452856 originate from mouse embryos and show base identity with the nucleotides (nt) from about 1060 to about 1450 and from about 24 to 440, respectively, of SEQ. ID NO. 1, or from about 1144 to about 1534 and from about 108 to about 524, respectively, according to the nucleotide positions in SEQ. ID NO. 3. Up until now no reading frame or function has been assigned to any of these 4 sequences.
The nucleotide sequence depicted in SEQ. ID NO. 1 contains an open reading frame which corresponds to a polypeptide having a length of 302 amino acids. This polypeptide is indicated in the amino acid sequence depicted SEQ. ID NO. 2. SEQ. ID NO. 3 shows a nucleotide sequence as in SEQ. ID NO. 1, but it has 84 additional nucleotides at the 5xe2x80x2 end. As a result, the positions of the nucleotides corresponding to each other shift by 84 nucleotides in each case. The polypeptide encoded by SEQ. ID NO. 3 therefore has 28 additional amino acids at the N terminus and is depicted in SEQ. ID NO. 4 with its total of 330 amino acids. SEQ. ID NO. 2 and 4 depict a C-terminal segment of the native polypeptide.
For illustration purposes reference is made to FIG. 1 which shows a diagrammatic representation of the cDNA of the endometriosis-associated gene according to the invention. Five exons, E1 to E5, and the position of fragment 1 (394 nt) used as a probe in DDRT-PCR are shown. The positions of the PCR primers (see example 4, table 1) used for RT-PCR are also shown.
Not shown in FIG. 1 is a further exon 4a whose nucleotide sequence is shown in SEQ. ID NO. 5. This exon 4a may be present. If it is present, it is found between exon 4 and exon 5. This corresponds to the position between nt1054 and nt1055 in SEQ. ID NO. 3. A combination of the sequences SEQ. ID NO. 1/3 with SEQ. ID NO. 5 is accordingly, for example, a sequence which contains the sequence of the exon 4a at said position.
Besides the nucleotide sequences shown in SEQ. ID NO. 1, 3 and 5 and combinations thereof such as the sequence of SEQ. ID NO. 3, which has the sequence of SEQ. ID NO. 5 between nt1054 and 1055 and to a nucleotide sequences which corresponds to the sequences within the scope of the degeneracy of the genetic code, the present invention also includes nucleotide sequences which hybridize with one of the sequences mentioned before. The term xe2x80x9chybridizationxe2x80x9d according to the present invention is used by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). Preferably a hybridization is called stringent if a positive hybridization signal is still observed after washing for one hour with 1xc3x97SSC and 0.1% SDS at 50xc2x0 C., preferably at 55xc2x0 C., particularly preferably at 62xc2x0 C. and most preferably at 68xc2x0 C., in particular for 1 h in 0.2xc3x97SSC and 0.1% SDS at 55xc2x0 C., preferably at 55xc2x0 C., particularly preferably at 62xc2x0 C. and most preferably at 68xc2x0 C. A nucleotide sequence hybridizing under these washing conditions with one or more of the nucleotide sequences depicted in SEQ ID NO. 1, 3 and 5, or with a nucleic sequence corresponding to these sequences within the scope of the degeneracy of the genetic code, is a nucleotide sequence according to the invention.
The nucleotide sequence according to the invention is preferably a DNA. However, it can also include an RNA or a nucleic acid analog such as a peptidic nucleic acid, for example. Particularly preferably the nucleic acid according to the invention includes a protein-encoding segment of the nucleotide sequences depicted in SEQ ID NO. 1, 3 and/or 5 or a sequence having a homology of more than 80%, preferably more than 90% and particularly preferably more than 95% to the nucleotide sequences depicted in SEQ ID NO. 1, 3 or 5 or a segment of preferably at least 20 nucleotides (nt) and particularly preferably at least 50 nt thereof. The same also holds for nucleic acids which have, as described above, the sequence of SEQ. ID NO. 5 in addition to those of SEQ ID NO. 1 or 3. The homology is given in percent identical positions when two nucleic acids (or peptide chains) are compared, where a 100% homology means complete identity of the compared chain molecules (Herder: Lexikon der Biochemie und Molekularbiologie [Dictionary of biochemistry and molecular biology], Spektrum Akademischer Verlag 1995).
Nucleic acids according to the invention are preferably obtainable from mammals and in particular from humans. They may be isolated according to known techniques by using short segments of the nucleotide sequences shown in SEQ. ID NO. 1, 3 or/and 5 as hybridization probes and/or as amplification primers. Furthermore, the nucleic acids according to the invention may also be prepared by chemical synthesis, it being possible to employ modified nucleotide building blocks, for example 2xe2x80x2-O-alkylated nucleotide building blocks, where appropriate, instead of conventional nucleotide building blocks.
The nucleic acids according to the invention or segments thereof may therefore be used for preparing primers and probes which preferably contain markers or labeling groups. Preference is also given to intron-bridging oligonucleotide primers which are particularly suitable for identifying different mRNA species.
The present invention further relates to polypeptides encoded by the nucleic acids defined as above. These polypeptides preferably comprise
(a) the amino acid sequence depicted in SEQ ID NO. 2 or 4 or
(b) a homology of more than 70%, preferably of more than 80% and particularly preferably of more than 90% to the amino acid sequence according to (a).
Besides the polypeptides depicted in SEQ ID NO. 2 or 4, the invention also relates to muteins, variants and fragments thereof. These are sequences which differ from the amino acid sequences depicted in SEQ ID NO. 2 or 4 by substitution, deletion and/or insertion of single amino acids or of short amino acid segments.
The term xe2x80x9cvariantxe2x80x9d includes both naturally occurring allelic variations or splicing variations of the endometriotic protein, and proteins generated by recombinant DNA technology (in particular in vitro mutagenesis with the aid of chemically synthesised oligonucleotides) which correspond substantially to the proteins depicted in SEQ ID NO. 2 or 4 with respect to their biological and/or immunological activity. This term also includes chemically modified polypeptides. Polypeptides which are modified at the termini and/or in the reactive amino acid side groups by acylation, for example acetylation or amidation belong to this group. Polypeptide fragments (peptides) representing a segment of at least 10 amino acids of the amino acid sequence shown in SEQ ID NO. 2 or 4 also belong to the amino acid sequences according to the invention.
The present invention further relates to a vector containing at least one copy of a nucleic acid according to the invention. This vector may be any prokaryotic or eukaryotic vector on which the DNA sequence according to the invention, preferably linked to expression signals such as promoter, operator, enhancer etc., is located. Examples of prokaryotic vectors are chromosomal vectors such as bacteriophages and extrachromosomal vectors such as plasmids, with circular plasmid vectors being particularly preferred. Suitable prokaryotic vectors are described, for example, in Sambrook et al., supra, Chapters 1-4. Particularly preferred is the vector according to the invention, a eukaryotic vector, e.g. a yeast vector, or a vector suitable for higher cells, e.g. plasmid vector, viral vector or plant vector. Vectors of this type are well known to the skilled worker in the field of molecular biology so that there is no need for further explanation here. In particular, reference is made in this connection to Sambrook et al., supra, Chapter 16.
The invention also relates to a vector which contains a segment of at least 21 nucleotides in length of the sequences depicted in SEQ ID NO. 1, 3 or/and 5 or a combination thereof. Preferably this segment has a nucleotide sequence which originates from the protein-encoding region of said sequences or from a region essential for the expression of the protein or polypeptide. These nucleic acids are particularly suitable for preparing therapeutically employable antisense nucleic acids preferably of up to 50 nucleotides in length.
The present invention further relates to a cell. transformed with a nucleic acid according to the invention or a vector according to the invention. The cell can be both a eukaryotic and a prokaryotic cell. Methods for transforming cells with nucleic acids are general prior art and therefore need no further explanation. Examples of preferred cells are eukaryotic cells, in particular animal and particularly preferably mammalian cells.
The present invention further relates to an antibody or a fragment of such an antibody against the polypeptide(s) encoded by the endometriosis gene or against variants thereof. Antibodies of this type are particularly preferably directed against complete polypeptides encoded by it or against a peptide sequence corresponding to amino acids 1-330 of the amino acid sequence depicted in SEQ ID NO. 4.
Identification, isolation and expression of a gene according to the invention which is specifically associated with invasive processes and in particular with endometriosis provide the requirements for diagnosis, therapy and prevention of diseases based on those disorders mentioned above.
It becomes possible with the aid of a polypeptide according to the invention or fragments of this polypeptide as immunogen to prepare antibodies against those polypeptides. Preparation of antibodies may be carried out in the usual way by immunizing experimental animals with the complete polypeptide or fragments thereof and subsequently obtaining the resulting polyclonal antisera. According to the method of Kxc3x6hler and Milstein and its developments monoclonal antibodies can be obtained from the antibody-producing cells of the experimental animals by cell fusion in the known manner. In the same way, human monoclonal antibodies can be produced according to known methods. Antibodies of this type could then be used both for diagnostic tests, in particular of endometriotic cell tissue, or else for the therapy.
For example, samples such as body fluids, in particular human body fluids (e.g. blood, lymph or CSF) may be tested with the aid of the ELISA technique on the one hand for the presence of a polypeptide encoded by the endometriosis gene, on the other hand for the presence of autoantibodies against such a polypeptide. Polypeptides encoded by the endometriosis gene or fragments thereof can then be detected in such samples with the aid of a specific antibody, for example of an antibody according to the invention. For detecting autoantibodies it is preferably possible to employ recombinant fusion proteins which contain a part or a domain or even the complete polypeptide encoded by the endometriosis gene and which are fused to a protein domain which facilitates detection, for example maltose-binding protein (MBP).
Diagnostic tests may also be carried out with the aid of specific nucleic acid probes for detecting at the nucleic acid level, for example at the gene or transcript level.
Provision of the nucleotide and amino acid sequences and antibodies according to the invention further facilitates a targeted search for effectors of the polypeptides/proteins. Effectors are agents which act in an inhibitory or activating manner on the polypeptide according to the invention and which are capable of selectively influencing cell functions controlled by the polypeptides. These may then be employed in the therapy of appropriate pathologies, such as those based on invasive processes. The invention therefore also relates to a method for identifying effectors of endometriotic proteins where cells expressing the protein are brought into contact with various potential effector substances, for example low molecular weight agents, and the cells are analyzed for modifications, for example cell-activating, cell-inhibiting, cell-proliferative and/or cell-genetic modifications. In this way it is also possible to identify binding targets of endometriotic proteins.
Since many neoplastic diseases are accompanied by invasive processes, the discovery of the gene according to the invention additionally provides possibilities for the diagnosis, prevention and therapy of cancerous diseases.
The discovery of a gene involved in the responsibility for invasive processes not only opens up possibilities for the treatment of diseases based on cellular modifications of this type, but the sequences according to the invention may also be used in order to make such processes usable. This can be of importance, for example, for the implantation of embryos.
The present invention therefore also relates to a pharmaceutical composition which includes as active components nucleic acids, vectors, cells, polypeptides, peptides and/or antibodies, as mentioned before.
The pharmaceutical composition according to the invention may further contain pharmaceutically conventional carriers, excipients and/or additives and, where appropriate, further active components. The pharmaceutical composition may be employed in particular for the diagnosis, therapy or prevention of diseases associated with invasive processes. Furthermore the composition according to the invention may also be employed for diagnosing a predisposition for such diseases, in particular for diagnosing an endometriosis risk.